We generated a novel model of mice heterozygous for a targeted null mutation of Tgfbr1. When crossed with mice carrying a mutation in the Apc tumor suppressor gene, these mice develop twice as many intestinal tumors as wild-type littermates. Invasive adenocarcinoma with features of human colon cancer is only identified among ApcMin/+; Tgfbr1+/- mice, not among ApcMin/+; Tgfbr1+/+ mice and tumors do not exhibit loss of heterozygosity at the Tgfbr1 locus. TGF-?-mediated growth inhibition, phosphorylation of Smad2 and Smad3, and overall TGF-? signaling are mildly decreased in haploinsufficient embryonic fibroblasts. Decreased Smad2 and Smad3 phosphorylation is observed in the colonic epithelium crypts of ApcMin/+; Tgfbr1+/- mice. Decreased Smad signaling is associated with increased cell proliferation in the crypts of the intestinal mucosa and intestinal tumors. Thus, constitutively reduced Tgfbr1-mediated signaling is a potent modifier of colorectal cancer development. To determine the relevance of these findings in humans, we analyzed germline peripheral blood for TGFBR1 expression. We found that 29 of 242 (12.0%) patients with colorectal cancer but only three of 195 (1.5%) controls had evidence of germline allele-specific expression of TGFBR1 (TGFBR1 ASE). These results indicate that TGFR1 ASE is one of the most commonly inherited cause of colorectal cancer, which increases cancer risk by approximately 870%. Differences in TGF-?-mediated phosphorylation of SMAD2 and SMAD3 between human lymphoid cells from individuals with wild-type TGFBR1 and individuals with TGFBR1 ASE were comparable to those found between Tgbr1+/+ and Tgfbr1+/- mouse embryonic fibroblasts. It is the purpose of the studies outlined in this application to unravel the molecular mechanisms of Tgfbr1 haploinsufficiency in colorectal cancer through execution of three Specific Aims: First: To characterize the phenotype of Tgfbr1+/+ and Tgfbr1+/- mice with respect to colon cancer: The phenotype of Tgfbr1+/+ and Tgfbr1+/- mice will be evaluated in three different backgrounds (129Sv/J, C57BL/6 and FVB/N). Colon tumors will be induced using the azoxymethane protocol and by crossing the transgenic strains with Apc1638N/+ and KRASV12G; Apc1638N/+ mice. Second: To determine the role of Tgfbr1 haploinsufficiency in development of cancer stem cells and intestinal stem cells as well as the contribution of stromal and lymphoepithelial Tgfbr1 signaling to tumor formation: We will determine the growth of Tgfbr1+/+ and Tgfbr1+/- tumors in nude mice to evaluate the contribution of Tgfbr1 haploinsufficiency on tumor stem cell development. Using the Lgr5 mouse model, we will also assess whether Tgfbr1 haploinsufficiency enhances the development of intestinal stem cells. The role of Tgfbr1 haploinsufficiency within stromal and lymphoepithelial cells will be evaluated as it relates to colon cancer development. Third: To assess Tgfbr1 haploinsufficiency oncogenic properties: Using MEFs from three distinct genetic backgrounds (129Sv/J, C57BL/6 and FVB/N) we will determine the extent to which Tgfbr1 haploinsufficiency enhances oncogenesis. PUBLIC HEALTH RELEVANCE: We have shown that decreased expression of the type I TGF-? receptor gene is emerging as the most common cause of colorectal cancer reported to date. The primary goal of this project is to understand the mechanisms linking decreased gene expression and colorectal cancer development. Experiments will be conducted with mice that only express one copy of the gene, which will be bred together with mice that spontaneously develop colon cancer.